Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-05-27
2002-08-13
Eyler, Yvonne (Department: 1646)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024310, C536S023100, C435S069100, C435S320100, C435S325000, C435S252300, C435S017000, C435S235100, C435S255200, C530S350000, C530S351000
Reexamination Certificate
active
06433154
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns processes for detection of ligand binding, as well as yeast cells, nucleic acids, vectors and fusion proteins useful therefor.
BACKGROUND OF THE INVENTION
In order to identify novel agonists and antagonists of mammalian receptor tyrosine kinases (RTK), we wished to express the extracellular domains (ECD) of these single transmembrane proteins in a microorganism. The yeast
Saccharomyces cerevisiae
is an extremely well-studied unicellular eukaryote for which a vast array of genetic and biochemical methodologies are available. Although yeast does not possess tyrosine kinase receptors, it has been shown to utilize numerous mitogen-activated protein kinase (MAPK) pathways that are activated by plasma membrane protein receptors. We chose one of these pathways, the HOG (high-osmolarity/glycerol) pathway, in an attempt to model RTK-ligand binding functions.
Yeast respond to high external osmolar conditions by increasing the intracellular concentration of glycerol. Varela et al. (1996),
Microbiology
142: 721-31. The HOG MAP kinase pathway in yeast (
FIG. 1
) consists of a histidine/aspartate/histidine/aspartate kinase relay coupled to a MAP kinase cascade. The SLN1 gene encodes the first histidine/aspartate kinases and functions as the plasma membrane sensor protein for the system. Under normal growth conditions, the Sln1 protein, a two-transmembrane (TM) plasma membrane kinase, is dimerized and actively transfers a phosphate to another protein, YPD1p, which in turn transfers the phosphate to another protein SSK1p. Posas et al. (1996),
Cell
86: 865-75. Under high osmolar conditions, however, the Sln1 protein is deactivated. The lack of phosphorelay through the histidine/aspartate (HDHD) pathway causes phosphorylation and activation of the HOG MAPK pathway. Unphosphorylated Ssk1 actively phosphorylates the MAPKKK's, Ssk2 and Ssk22, which transfer the phosphate to the MAPKK, Pbs2, which in turn transfers the phosphate to the MAPK, Hog1. Phosphorylated Hog1 has been shown to activate transcription factors which increase production of enzymes involved in glycerol synthesis and stress response. Schuller et al. (1994),
EMBO I
13: 4382-9.
SUMMARY OF THE INVENTION
By substituting the ECD of receptor tyrosine kinases (RTK) for that of such sensors as Sln1 protein, we model the ligand binding function of the ligand-binding proteins in yeast. This invention thus concerns
Saccharomyces cerevisiae
cells comprising a fusion protein having:
(a) an extracellular ligand-binding domain derived from a protein of interest, and
(b) an intracellular kinase domain that is activated upon binding of a ligand;
wherein binding of a ligand is signaled by transfer of a phosphate by the intracellular domain. The invention also concerns the nucleic acid encoding the fusion protein, the associated vector, and the fusion protein itself.
The invention also concerns a process for detecting binding of a ligand to a protein of interest, which comprises:
(a) treating a culture of such cells with a test substance, and
(b) detecting activation of the intracellular kinase domain.
One manner in which the detecting step may be carried out is by the detection of phosphorylation of Hog1, a method for which is described in detail hereinafter.
In preferred embodiments, the intracellular kinase domain is derived from SLN1; the extracellular ligand binding domain from a mammalian receptor tyrosine kinase. In a preferred application of the invention, the extracellular ligand binding domain is derived from human epidermal growth factor receptor. The preferred
Saccharomyces
cells are strain SDO158 (BMS Culture Collection Accession Number SGY1661).
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Gorman Jessica A.
Ostrander Darin B.
Basi Nirmal S.
Bristol--Myers Squibb Company
Eyler Yvonne
Wilk-Orescan Rosemarie R.
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